Primer reporte de la obtención de mutantes del pez Cebra (Danio rerio) por pérdida de pigmentación de la piel por la edición génica del gen de la tirosinasa con el sistema Crispr/Cas9
DOI:
https://doi.org/10.57188/Palavras-chave:
Crispr Cas, edición génica, pez Cebra, pigmentación, tirosinasaResumo
Actualmente el sistema Crispr/Cas9 permite la edición del genoma en forma eficiente para inducir específicamente mutaciones deseadas. En este estudio, se describe la inducción de mutaciones del gen de la tirosinasa (Tyr) en el pez Cebra para probar la efectividad de la microinyección. Una secuencia de un ARN guía (gRNAs) de 20 nucleótidos y dos primers flanqueadores (forward y reverse) fueron diseñados para el gen de la tirosinasa y microinyectados en los embriones post fecundados y mostraron una pérdida gradual de pigmentación a nivel corporal desde las primeras etapas embrionarias hasta la adultez. Este estudio reporta por primera en el Perú que el sistema Crispr/Cas9 puede realizarse en el pez Cebra como animal modelo de entrenamiento para modificar el gen Tyr cuyos fenotipos despigmentados fueron fácilmente distinguibles evidenciándose su éxito en la edición génica. A futuro serviría como modelo bioexperimental para producirse mutaciones de interés de otros genes de otras especies acuícolas de alto valor ornamental y/o comercial para el Perú.
Downloads
Referências
Aulia, A., Hutapea, R., Setya Abdima, P., Ali Emawan, A., & Edbert, I. (2023). CRISPR: On How it’ll Change the Future. Engineering, Mathematics and Computer Science, 5(2), 73-77. https://doi.org/10.21512/emacsjournal.v5i2.9975
Altschul, S. F., Gish, W., Miller, W., Myers, E. W., & Lipman, D. J. (1990). Basic local alignment search tool. Journal of Molecular Biology, 215, 403-410. https://doi.org/10.1016/S0022-2836(05)80360-2
Base de datos del NCBI. https://www.ncbi.nlm.nih.gov/
Basolo, A. (2006). Genetic Linkage and Color Polymorphism in the Southern Platyfish (Xiphophorus maculatus): A Model System for Studies of Color Pattern Evolution. Zebrafish, 3(1), 65-83. https://doi.org/10.1089/zeb.2006.3.65
Bell, C. C., Magor, G. W., Gillinder, K. R., & Perkins, A. C. (2014). A high-throughput screening strategy for detecting CRISPR-Cas9 induced mutations using next-generation sequencing. BMC Genomics, 15, 1002. https://doi.org/10.1186/1471-2164-15-1002
Bian, C., Li, R., Wen, Z., Ge, W., & Shi, Q. (2021). Phylogenetic analysis of core melanin synthesis genes provides novel insights into the molecular basis of albinism in fish. Frontiers in Genetics, 12, 707228. https://doi.org/10.3389/fgene.2021.707228
Boonanuntanasarn, S., Yoshizaki, G., Iwai, K., & Takeuchi, T. (2004). Molecular cloning, gene expression in albino mutants and gene knockdown studies of tyrosinase mRNA in rainbow trout. Pigment Cell Research, 17(4), 413-21. https://doi.org/10.1111/j.1600-0749.2004.00166.x
Brinkman, E., Chen, T., de Haas, M., Holland, H., Akhtar, W., & Steensel, B. (2018). Kinetics and fidelity of the repair of Cas9-Induced Double-Strand DNA breaks. Molecular Cell, 70, 801–813. https://doi.org/10.1016/j
Braasch, I.; Schartl, M., & Volff, J. (2007). Evolution of pigment synthesis pathways by gene and genome duplication in fish. BMC Evolutionary Biology, 7(74), 14-18. https://doi.org/10.1186/1471-2148-7-74
Camp, E., Badhwar, P., Mann, G., & Lardelli, M. (2003). Expression analysis of a tyrosinase promoter sequence in zebrafish. Pigment Cell Research, 16, 117–126. https://doi.org/10.1034/j.1600-0749.2003.00002.x
Chang, N., Sun, C., Gao, L. et al. (2013). Genome editing with RNA-guided Cas9 nuclease in zebrafish embryos. Cell Research, 23, 465–472. https://doi.org/10.1038/cr.2013.45
Changqing, Z., Ziheng, R., & Zhiyuan, G. (2023). Generation of Albino Phenotype in Ornamental Fish by CRISPR/Cas9-Mediated Genome Editing of slc45a2 Gene. Marine Biotechnology, 25, 1-10. https://doi.org/10.1007/s10126-023-10204-9
Chaudhary, D. K., Singh, S. K., Gohil, N., & Bhattacharjee, G. (2020). Recent progress of CRISPR-Cas9 in zebra fish in Genome engineering via CRISPR/cas9 system. In: Genome Engineering via CRISPR-Cas9 System. Chapter 19, Editorial: Elsevier Inc., 251-261. https://doi.org/10.1016/B978-0-12-818140-9.00019-2
Dooley, CM, Schwarz, H., Mueller, K.P., Mongera, A., Konantz, M.; Neuhauss, S., Nusslein-Volhard, C., & Geisler, R. (2012). Slc45a2 and V-ATPase are regulators of melanosomal pH homeostasis in zebrafish, providing a mechanism for human pigment evolution and disease. Pigment Cell Melanoma Research, 26, 205–217. https://doi.org/10.1111/pcmr.12053
Fan, Y., Zhang, G., Zhao, K., Yuan, X., Fu, W., Liu, J., Liu, W., Peng, L., & Xiao, Y. (2023). Rapidly generating homozygous mutate zebrafish in F0 generation by technical integration of CRISPR/Cas9 and gynogenesis. Reproduction and Breeding, 3(2), 45-49. https://doi.org/10.1016/j.repbre.2023.04.001
Food and Agriculture Organization. (2020). FAO Aquaculture Newsletter No. 61. http://www.fao.org/faoterm/collection/aquaculture/en/
Ferdous, Md. A., Islam, S. I., Habib, N., Almohada, M., Allahyani, M., Alsaiari, A. A., & Shafie, A. (2022). CRISPR-Cas Genome Editing Technique for Fish Disease Management: Current Study and Future Perspective. Microorganisms, 10(10), 2012. https://doi.org/10.3390/microorganisms10102012
Fujii, R. (2000). The regulation of motile activity in fish chromatophores. Pigment cell research, 13, 300-319. https://doi.org/10.1034/j.1600-0749.2000.130502.x
Genbank (2023a). Secuencia del ARNm del gen Tyr parcial de Danio rerio para la enzima tirosinasa NM_131013.3. https://www.ncbi.nlm.nih.gov/nuccore/NM_131013
Genbank (2023b). Gen tyr parcial de Danio rerio para la enzima tirosinasa XM_003451484.3. https://www.ncbi.nlm.nih.gov/nucleotide/AJ489318.1?report=genbank&log$=nucltop&blast_rank=58&RID=UXN4KZ8M013
Grainger, S., Lonquich, B., Oon, C. H., Nguyen, N., Willert, K., & Traver, D. (2017). CRISPR guide RNA validation in vitro. Zebrafish 14, 383–386. https://doi.org/10.1089/zeb.2016.1358
Gutási, A., Hammer, S.E., El-Matbouli, M., & Saleh, M. (2023). Review: Recent Applications of Gene Editing in Fish Species and Aquatic Medicine. Animals, 13, 1250. https://doi.org/10.3390/ani13071250
Hallerman, E. (2021). Genome Editing in Cultured Fishes. CABI Agricultura y Biociencia, 2. https://doi.org/10.1186/s43170-021-00066-3
Han, J., Kraft, P., Nan, H., Guo, H., Chen, C., Qureshi, A., Hankinson, E., Hu, F., Duffy, D., Zhao, Z., Martin, N., Montgomery, G., Hayward, N., & Hunter, A. (2008). Genome-Wide Association Study Identifies Novel Alleles Associated with Hair Color and Skin Pigmentation. PLoS Genet, 4(5), 1-11. https://doi.org/10.1371/journal.pgen.1000074
Hisano, Y., Ota, S., & Kawahara, A. (2014). Genome editing using artificial site-specific nucleases in zebrafish. Development, Growth & Differentiation, 56(1):26-33. https://doi.org/10.1111/dgd.12094
Hruscha, A., Krawits, P., Rechenberg, A., Heinrich, V., Hecht, J., Haass, C., & Schmid, B. (2013). Efficient CRISPR/Cas9 genome editing with low off-target effects in zebrafish. Development, 140, 4982–4987. https://doi.org/10.1242/dev.099085
Hwang, W.Y., Fu, Y., Reyon, D., Maeder, M. L., Kaini, P., et al. (2013) Heritable and precise zebrafish genome editing using a CRISPR-Cas System. PLoS ONE, 8(7), e68708. https://doi.org/10.1371/journal.pone.0068708
Iida, A., Inagaki, H., Suzuki, M., Wakamatsu, Y., & Hori, H. (2004). The tyrosinase gene of the i(b) albino mutant of the medaka Fish carries a transposable element insertion in the promoter. Pigment Cell Research, 17, 158–164. https://doi.org/10.1046/j.1600-0749.2003.00122.x
Inagaki, H., Koga, A., Bessho, Y., & Hori, H. (1998). The tyrosinase gene from medakafish: transgenic expression rescues albino mutation. Pigment Cell Research, 11(5), 283-90. https://doi.org/10.1111/j.1600-0749.1998.tb00737.x
Jao, L. E., Wente, S. R., & Chen, W. (2013) Efficient multiplex biallelic zebrafish genome editing using a CRISPR nuclease system. Proceedings of the National Academy of Sciences USA, 110, 13904–13909. https://doi.org/10.1073/pnas.1308335110
Jinek, M., Chylinski, K., Fonfara, I., Hauer, M., Doudna, J. A., & Charpentier, E. A (2012). Programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science, 337(6096), 816-21. https://doi.org/10.1126/science.1225829
Kelsh, R. N. Genetics and evolution of pigment patterns in fish. (2004). Pigment Cell Research, 17, 326–336. https://doi.org/10.1111/j.1600-0749.2004.00174.x
Kimmel, C. B., Ballard, W. W., Kimmel, S. R., Ullmann, B., & Schilling, T. F. (1995). Stages of embryonic development of the zebrafish. Developmental Dynamics, 203(3), 253-310. https://doi.org/10.1002/aja.1002030302
Kroll, F., Powell, G., Ghosh, M., Gestri, G., Antinucci, P., Hearn, T., Tunbak, H., Lim, S., Dennis, H., Fernandez, J., Whitmore, D., Dreosti, E., Wilson, S., Hoffman, E., & Rihel, J. (2021). A simple and effective F0 knockout method for rapid screening of behaviour and other complex phenotypes. eLife, 10, e59683. https://doi.org/10.7554/eLife.59683
Krug, J., Perner, B., Albertz, C., Mörl, H., Hopfenmüller, V., Englert, C. (2023) Generation of a transparent killifish line through multiplex CRISPR/Cas9mediated gene inactivation. eLife, 12, e81549. https://doi.org/10.7554/eLife.81549
Ley que establece la moratoria al ingreso y producción de organismos vivos modificados al territorio nacional por un período de 10 años (Ley Nº 29811, 2011, Diciembre 09). Diario Oficial El Peruano, pp. 454601. En: https://www.minam.gob.pe/wp-content/uploads/2017/04/Ley-N%C2%B0-29811.pdf.
Leído 03 de diciembre de 2025.
Ley que modifica la Ley 29811, Ley que establece la moratoria al ingreso y producción de organismos vivos modificados al territorio nacional por un período de 15 años, a fin de establecer la moratoria hasta el 31 de diciembre de 2035 (Ley Nº 31111, 2021, Enero 06). Diario Oficial El Peruano, pp. 4. En: https://bioseguridad.minam.gob.pe/wp-content/uploads/2021/02/Ley-31111.pdf
Liu, J., Gong, L., Chang, C., Liu, C., Peng, J., & Chen, J. (2012). Development of novel visual plus quantitative analysis systems for studying DNA double-strand break repairs in zebrafish. Journal of Genetics and Genomics, 39(9), 489-502. https://doi.org/10.1016/j.jgg.2012.07.009
Mojica, F. J. M., Díez-Villaseñor, C., García-Martínez, J., & Almendros, C. (2009). Short motif sequences determine the targets of the prokaryotic CRISPR defence system. Microbiology (Reading), 155(Pt 3), 733-740. https://doi.org/ 10.1099/mic.0.023960-0
Moreno-Mateos, M., Vejnar, C., Beaudoin, J. D., et al. (2015). CRISPRscan: designing highly efficient sgRNAs for CRISPR-Cas9 targeting in vivo. Nature Methods, 12, 982–988. https://doi.org/10.1038/nmeth.3543
Okoli, A., Blix, T., Myhr, A., Xu, W., & Xu, X. (2022). Sustainable use of CRISPR/Cas in fish aquaculture: the biosafety perspective. Transgenic Research, 31(1), 1-21. https://doi.org/10.1007/s11248-021-00274-7
Page-McCaw, P. S., Chung, S. C., Muto, A., Roeser, T., Staub, W., Finger-Baier, K. C., Korenbrot, J. I., & Baier, H. (2004). Retinal network adaptation to bright light requires tyrosinase. Nature Neuroscience, 7(12), 1329-1336. https://doi.org/10.1038/nn1344
Parichy, D. M. (2006). Evolution of danio pigment pattern development. Heredity (Edinb). 97, 200–210. https://doi.org/10.1038/sj.hdy.6800867
Preeti, S., Sharan, S., & Ramtej, V. (2021). CRISPR-based genome editing of zebrafish. Progress in Molecular Biology and Translational Science, 180, 69-84. https://doi.org/10.1016/bs.pmbts.2021.01.005
Programa Primer-BLAST. https://www.ncbi.nlm.nih.gov/tools/primer-blast/
Programa OligoAnalyzer Tool. https://www.idtdna.com/calc/analyzer
Proyecto de Ley 011125/2024-CR, que busca promover y regular el uso de variedades y razas biotecnológicas en agricultura y ganadería, fomentando investigación, innovación y la sobera-nía genética incluyendo Organismos Vivos Modificados (OVM) y Organismos Genéticamente Editados (OGE). (Proyecto de Ley Nº 011125, 2025, Mayo 13). Congreso de la República.
Puthumana, J., Chandrababu, A., Sarasan, M., et al. (2024). Genetic improvement in edible fish: status, constraints, and prospects on CRISPR-based genome engineering. Biotech, 14, 44. https://doi.org/10.1007/s13205-023-03891-7
Roy, S., Kumar, V., Behera, B. K., Parhi, J., Mohapatra, S., Chakraborty, T., & Das, B. K. (2022). CRISPR/Cas Genome Editing—Can It Become a Game Changer in Future Fisheries Sector? Frontiers in Marine Science, 9, artículo 924475. https://doi.org/10.3389/fmars.2022.924475
Shiraki, T., & Kawakami, K. (2024). Generation of Transgenic Fish Harboring CRISPR/Cas9-Mediated Somatic Mutations Via a tRNA-Based Multiplex sgRNA Expression. Methods in Molecular Biology, 2707, 305-318. https://doi.org/10.1007/978-1-0716-3401-1_20
Sorlien, E., Witucki, M. A., & Ogas, J. (2018). Efficient Production and Identification of CRISPR/Cas9-generated Gene Knockouts in the Model System Danio rerio. Journal of Visualized Experiments, 138, 56969. https://doi.org/10.3791/56969
Sung, Y. H., Kim, J. M., Kim, H. T., Lee, J., Jeon, J., Jin, Y., Choi, J. H., Ban, Y. H., Ha, S. J., Kim, C. H., Lee, H. W., & Kim, J. S. (2014). Highly efficient gene knockout in mice and zebrafish with RNA-guided endonucleases. Genome Research, 24, 125–131. https://doi.org/10.1101/gr.163394.113
Ota, S., Hisano, Y., Ikawa, Y., & Kawahara, A. (2014). Multiple genome modifications by the CRISPR/Cas9 system in zebrafish. Genes Cells, 19(7), 555-564. https://doi.org/10.1111/gtc.12154
Tsetskhladze, Z. R., Canfield, V. A., Ang, K. C., Wentzel, S. M., Reid, K. P., Berg, A. S., Johnson, S. L., Kawakami, K., & Cheng, K. C. (2012). Functional assessment of human coding mutations affecting skin pigmentation using zebrafish. PLoS One, 7(10), e47398. https://doi.org/10.1371/journal.pone.0047398
Untergasser. A., Cutcutache, I., Koressaar, T., Ye, J., Faircloth, B. C., Remm, M., & Rozen, S. G. (2012). Primer3 - new capabilities and interfaces. Nucleic Acids Research, 40(15), e115. https://doi.org/10.1093/nar/gks596
Varshney, G., Sood, R., & Burgess, S. (2015). Understanding and Editing the Zebrafish Genome. Advanced Genetics, 92, 1-52. https://doi.org/10.1016/bs.adgen.2015.09.002
Wang, J., Hou, L., Zhang, R. et al. (2007). The tyrosinase gene family and albinism in fish. Journal of Oceanology and Limnology, 25, 191–198. https://doi.org/10.1007/s00343-007-0191-9
Westerfield, M. (2000). The Zebrafish Book: A Guide for the Laboratory Use of Zebrafish. http://zfin.org/zf_info/zfbook/zfbk.html.
White, R., Sessa, A., Burke, C., Bowman, T., LeBlanc, J., Ceol, C., Bourque, C., Dovey, M., Goessling, W., Burns. C., & Zon, L. (2008). Transparent adult zebrafish as a tool for in vivo transplantation analysis. Cell Stem Cell, 2, 183-189. https://doi.org/10.1016/j.stem.2007.11.002
Wu, Y., & Wang, I. (2020). Heat-shock-induced tyrosinase gene ablation with CRISPR in zebrafish. Molecular Genetics and Genomic, 295, 911–922. https://doi.org/10.1007/s00438-020-01681-x
Xu, X., Chen, H., Mandal, B. K., Si, Z., Wang, J., & Wang, C. (2022). Duplicated Tyr disruption using CRISPR/Cas9 reveals melanophore formation in Oujiang color common carp (Cyprinus carpio var. color). Reproduction and Breeding, 2, 37-45. https://doi.org/10.1016/j.repbre.2022.05.001
Yin, L., Maddison, L. Li, M., Kara, N., LaFave, M., Varshney, G., Burgess, S., Patton, J., & Chen, W. (2015). Multiplex Conditional Mutagenesis Using Transgenic Expression of Cas9 and sgRNA. Genetics, 200(2), 431–441. https://doi.org/10.1534/genetics.115.176917
Yu, C., Zhang, Y., Yao, S., & Y. Wei. (2014). A PCR based protocol for detecting indel mutations induced by TALENs and CRISPR/Cas9 in zebrafish. PLoS ONE, 9, e98282. https://doi.org/10.1371/journal.pone.0098282
Yu, L., Chen, H., Hu, X., Chen, X., Liu, Z., Wang, J., & Wang, C. (2021). SLC24A5 plays fundamental roles in regulating melanophore development in Cyprinidae fish. Reproduction and Breeding, 1, 167-173. https://doi.org/10.1016/j.repbre.2021.11.001
Yuan S., & Sun Z. (2009). Microinjection of mRNA and morpholino antisense oligonucleotides in zebrafish embryos. Journal of Visualized Experiments, 7(27), 1113. https://doi.org/10.3791/1113
Zebrafish Information Network (ZFIN) (2024). https://zfin.org/ZDB-FISH-1509017721
Zhang, Y., Qin, W., Lu, X., Xu, J., Huang, H., & Bai, H. (2017). Programmable base editing of zebrafish genome using a modified CRISPR-Cas9 system. Nature Communications, 8(1), 118. https://doi.org/10.1038/s41467-017-00175-6
Zhang, C., Ren, Z., & Gong, Z. (2023). Generation of Albino Phenotype in Ornamental Fish by CRISPR/Cas9-Mediated Genome Editing of slc45a2 Gene. Mar Biotechnol (NY), 25, 281-290. https://doi.org/10.1007/s10126-023-10204-9
Zhu, B., & Ge, W. (2018). Genome editing in fishes and their applications. General and Comparative Endocrinology, 257, 3-12. https://doi.org/10.1016/j.ygcen.2017.09.011
Downloads
Publicado
Edição
Secção
Licença
Direitos de Autor (c) 2025 Carlos Scotto
Este trabalho encontra-se publicado com a Licença Internacional Creative Commons Atribuição 4.0.
Manglar is an open access journal distributed under the terms and conditions of Creative Commons Attribution 4.0 International license
